Universal influenza vaccine

ABSTRACT

Immunogenic compositions for inducing a universal immune response to influenza, and particularly influenza A, by eliciting anti-neuraminidase antibodies which provide protection against heterologous influenza infection. Compositions comprising recombinant baculovirus expression vectors expressing neuraminidase in cultured insect cells dispersed in a pharmaceutically-acceptable carrier comprising insect cell culture media, and optional adjuvant. Methods of inducing immune responses against influenza, and particularly influenza A, by eliciting anti-neuraminidase antibodies in a host animal susceptible to infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/711,873,filed Dec. 12, 2019, which claims the priority benefit of U.S.Provisional Patent Application Ser. No. 62/778,409, filed Dec. 12, 2018,entitled UNIVERSAL INFLUENZA VACCINE, incorporated by reference in itsentirety herein.

SEQUENCE LISTING

The following application contains a sequence listing in computerreadable format (CRF), submitted as a text file in ASCII format entitled“Sequence Listing,” created on May 11, 2022, as 31,309 bytes. Thecontent of the CRF is hereby incorporated by reference.

BACKGROUND Field of the Invention

The present disclosure relates to vaccines against influenza, andparticularly influenza A, which elicit anti-neuraminidase antibodies.

Description of Related Art

Swine influenza (SI) is an acute respiratory disease caused by influenzaA viruses, which is an enveloped virus of the Orthomyxoviridae family.Swine influenza virus (SIV) is both a pathogen of economic significanceto the swine industry. An additional concern regarding SIV is itszoonotic potential as under certain circumstances it can be transmittedto humans, which is best exemplified by the 2009 H1N1 pandemicoriginated from a swine source. For that reason, it is a high priorityagent in animal health for the USDA.

A typical outbreak of respiratory disease caused by SIV is characterizedby sudden onset and rapid spread within a herd. Clinical symptomsassociated with SI may include coughing, sneezing, nasal discharge,elevated rectal temperature, lethargy, breathing difficulty, anddepressed appetite. While morbidity rates may reach 100% with SIVinfections, mortality rates are generally low.

The genome of SIV is separated into 8 independent RNA segments thatallows for frequent reassortment when two different viruses infect andreplicate within the same cell of a pig. Reassortment often results inthe production of a new influenza virus (antigenic shift), which rendersthe current strain-specific vaccine strategy ineffective. In addition,influenza A virus has the unique capacity to undergo genetic variations(antigenic drift) in that it can mutate up to 50% of the amino acidsequence of its major surface protein, hemagglutinin (HA), withoutchanging the function of the HA protein. Antigenic drift and antigenicshift contribute to apparent failures of SI vaccines when used in theswine industry.

In the USA, H1N1, H1N2 and H3N2 subtypes are the major causes of SIoutbreaks, although other subtypes have been isolated from diseasedpigs. Each subtype also consists of several genetic and antigenicclusters. The contemporary H3N2 SIV strain is a triple reassortantstrain, resulting from contributions of human, avian, and SI virallineages. H3N2 is widely spread in U.S. swine herds. A similarreassortment mechanism resulted in the emergence and rapid spread of atriple reassortant H1N1 virus that has also become endemic in U.S. swinepopulations.

Currently available SI vaccines are based on killed viruses containingH1N1, H1N2 and H3N2 subtypes. Due to antigenic drift and shift, thekilled vaccines are very limited in offering protection against SIVstrains genetically and antigenically different from the vaccinestrains. These killed vaccines appear also to be associated with diseaseenhancement in vaccinated pigs under some circumstances after virusinfection.

Nevertheless, vaccination remains the most effective approach to preventinfluenza epidemics. Conventional influenza vaccines can preventclinical disease, but their efficacy depends on the extent of antigenic“match” between the strains used for vaccine preparation and thosecirculating in the population. This challenge has focused numerousefforts towards discovering a vaccine candidate that elicits abroad-spectrum protection against diverse influenza subtypes, alsotermed a universal vaccine.

Influenza A virus has two large surface glycoproteins, HA andneuraminidase (NA). The HA mediates the viral receptor binding and entryprocess, while the NA catalytically drives the release of newly formedvirus particles as well as promotes the movement of virus particles viamucosal surfaces.

While several commercial inactivated whole-virus, as well as autogenousvaccines, are widely used, efficacy in field conditions is often lackingdue to genetic mismatch between vaccine strains and challenge virus. Theimmune response following vaccination is largely humoral and directedalmost exclusively against the immunodominant HA gene. While antibodiesrecognizing the HA are often neutralizing and can confer sterilizingimmunity, mismatch between antibodies and virus can lead to a loss ofbinding or even enhancement of disease, a phenomenon referred tovaccination associated-enhancement of respiratory disease (VAERD).

Numerous studies have shown that NA-specific antibodies are effective inoffering protection from influenza by blocking viral particle release.NA as an important target for control, and prevention of influenza Avirus infection has been demonstrated by FDA-approved therapeuticantiviral drugs like Oseltamivir and Zanamivir (NA inhibitors). Theviral NA differs from viral HA in that the NA protein is relatively moreconserved than HA among different influenza strains and swine and humanisolates principally consist of two major subtypes, termed N1 and N2.Currently marketed influenza vaccines contain an NA component (amongother viral particles) but are generally incapable of inducingprotective anti-NA antibodies largely due to the immunodominance of theviral HA protein that is also contained in such vaccines. Despite beinga viral surface glycoprotein like HA, the natural immune responsedirected towards NA is substantially less than that of HA. As aconsequence, NA exhibits less variability than HA and represents a moreconserved antigen. More prominent presentation of NA to the immunesystem, either by dissociation of the virion or by recombinantexpression of NA, has been shown to elicit a stronger anti-NA antibodyresponse.

Both the HA and NA proteins are critical to the influenza virus lifecycle. HA allows the virus to bind to cell surface sialic acids presenton cellular membrane proteins, leading to endocytosis and virusinternalization. In contrast, NA cleaves sialic acids from the host cellallowing mature virions to release and disseminate. While HA antibodiescan ideally prevent infection, NA antibodies allow cell infection butprevent virus dissemination. This allows for a more robust immuneresponse as antigen can be processed via both MHCI and MHCII,effectively stimulating infection immunity.

The baculovirus expression system is widely used for vaccine production.Examples in humans include vaccines for human papillomavirus andinfluenza virus, while in pigs baculovirus is used for numerous porcinecircovirus type 2 vaccines. Baculovirus expression is popular forvaccine antigen production owing to several features. Baculoviruses canonly infect certain insects, making them extremely safe for use inmammals. Large amounts of antigen can also easily be produced usingbaculovirus expression systems, making them economical.

SUMMARY OF INVENTION

Immunogenic compositions for inducing a universal immune response toinfluenza. The composition comprises recombinant baculovirus expressionvectors in cultured insect cells dispersed in apharmaceutically-acceptable carrier comprising insect cell culture mediaand optional adjuvant. The recombinant baculovirus expression vectorsexpress NA. Preferably, the NA is a recombinantly-expressed wild-type NAprotein.

Kits for stimulating a universal immune response against influenzainfection are also described. The kits comprise an immunogeniccomposition according to any one of the embodiments described herein andinstructions for administering the composition to a host animalsusceptible to influenza.

Also described herein are methods of stimulating a universal immuneresponse against influenza infection. The methods comprise administeringan immunogenic composition according to any one of the embodimentsdescribed herein to a host animal susceptible to influenza in aneffective amount. Advantageously, the immunogenic compositions provide a“universal” immune response in that the anti-NA antibodies stimulated bythe vaccine provide protection against heterologous influenza infections(i.e., infections from other strains which are different from the strainfrom which the recombinantly-expressed NA is derived).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating activity of NA expressed in therecombinant baculovirus expression system.

FIG. 2 is a graph showing the IDEXX IAV-S NP ELISA and NA inhibition(NI) titers in sera collected from pigs on the day of challenge.

FIG. 3 is a graph of the qRT-PCR Ct values for Ct values in nasal swabscollected from pigs on days 1, 3 and 5 post-challenge.

FIG. 4 is a graph of the SIV titers (TCID50/mL) in nasal swabs collectedfrom pigs on days 1, 3 and 5 post challenge.

FIG. 5 is a graph of qRT-PCR Ct values and percent lung consolidationday 5 post challenge.

FIG. 6 is a graph of the pathology scores from lung tissue staining.

FIG. 7 is a graph of the results of the NA inhibition assay showingantibody generated to N1 and N2.

FIG. 8 is a graph of PCR results from lung tissue collected 5 dayspost-challenge with heterologous H1N1.

FIG. 9 is graph of gross and microscopic pathology scores on lungscollected 5 days post challenge.

FIG. 10 shows images from SDS-PAGE and Western blotting of purified NA1and NA2 proteins.

FIG. 11 is a graph of Serology-NA inhibition (NI) antibody titer toparental viruses.

FIG. 12 is a graph of IAV PCR (Ct) values on nasal swabs collected ondays 1, 2 and 5 post challenge and lung tissue collected day 5 postchallenge.

FIG. 13 is a graph of gross and microscopic scores on lung tissuecollected day 5 post challenge.

FIG. 14 is a graph of Serology-NA inhibition (NI) antibody titer toparental viruses.

FIG. 15 is a graph of IAV PCR (Ct) values on nasal swabs collected ondays 3 and 5 post challenge and lung tissue collected day 5 postchallenge.

FIG. 16 a graph of gross and microscopic scores on lung tissue collectedday 5 post challenge.

FIG. 17 is a graph of Serology-NA inhibition (NI) antibody titer toparental viruses for diluted vaccine formulations.

FIG. 18 shows graphs scoring gross lung lesions after challenge for pigsvaccinated with diluted vaccine formulations.

FIG. 19 shows a graph of IHC and H&E staining on lung tissue from pigsvaccinated with diluted vaccine formulations.

DETAILED DESCRIPTION

Our universal influenza vaccine strategy is to develop arecombinantly-expressed neuraminidase-based protective vaccine forinfluenza virus, such as against SI, produced in a baculovirusexpression system. Baculovirus expression systems and baculovirusexpression vectors in general have been described extensively in theliterature, including U.S. Pat. No. 4,745,051, O'Reilly at al.(Baculovirus Expression Vectors, A Laboratory Manual. (1993)), andMurhammer (Baculovirus and Insect Cell Expression Protocols. In: Methodsin Molecular Biology™. Volume 388 (2007)), incorporated by referenceherein.

Immunogenic compositions are described herein which comprise recombinantbaculovirus expression vectors in cultured insect cells dispersed incell culture media, along with an optional adjuvant (oil-in-water,water-in-oil, etc.). The baculovirus expression vectors direct theexpression of native or wild-type NA proteins in the infected insectcells. That is, unlike previous approaches using modified NA proteins,such as those in which the transmembrane domain has been modified ordeleted, the present expression systems are engineered in a way that thefull-length, wild-type or native NA protein sequence is expressed in thesystem (and not a truncated version), and more particularly the nativeor wild-type NA protein is expressed as part of the insect cell membraneproteins, and can be found integrated into the insect cell membranes, aswell as in the budded baculovirus membranes in the culture supernatants.Preferably, the full-length, wild-type/native NA coding sequence (cDNA)is cloned into the baculovirus system for expression. It will beappreciated that such sequences can be identified based upon thecirculating strains isolated in a population at a given time, andsynthesized to create updated vaccines targeted to circulating strains.However, as shown in the data, the inventive vaccines provide protectionagainst heterologous challenge. Therefore, protective immunity may beobtained even from vaccines different from the circulating strains.Advantageously, since the expression systems are engineered to expressthe full-length, wild-type/native NA protein, which is associated withand presented by membrane components in the expression system (i.e., theinfected insect cells and the baculoviral components), and not as free,soluble recombinant NA in solution, it is believed that the expressed NAprotein in the inventive vaccine is presented to the immune system in amanner (folded structure) more closely resembling (and perhapssubstantially similar to) its native conformation, further enhancing theimmune response.

Crude and unpurified cultures of the expression system components arepreferably used for the immunogenic compositions, that is, as a crudecell culture containing the unpurified infected cells (presenting NA),baculovirus (expressing NA), and cell media, along with optionaladjuvant. In other words, the infected cell culture may be chemicallyinactivated, but is not otherwise processed or purified.

Exemplary insect cells for use in the invention include Lepidopteranspecies Spodoptera frugiperda and cell lines derived therefrom. Otherinsect cell hosts can be used including Trichoplusia ni, Bombyx mori,cell lines derived therefrom, and the like. Particularly preferredinsect cell lines include SF9 (and variants), SF21, High Five(BTI-TN-5B1-4), and the like. Any suitable culture media can be used topropagate the cells. Culture media suitable for insect cell culture ispreferably serum-free, and various formulations are known in the art andwidely available, such as SF900II. In general, the culture medium willcomprise a mixture of amino acids, sugars, salts, proteins, and thelike. Publicly available formulations such as Grace's media are alsosuitable. Various baculovirus expression systems are commerciallyavailable. Exemplary baculovirus expression vectors will generallyinclude ProEasy (AB Vector), BaculoGold™ DNA (PharMingen), Bac-N-Blue™DNA (Invitrogen), or BacPAK6™ DNA (Clontech) for co-transfection withthe transfer vector (donor or shuttle) plasmid DNA containing theforeign gene. Alternatively, insect cells are transfected with arecombinant bacmid DNA constructed by transposition of the donor plasmidDNA in E. coli cells, using the Bac-to-Bac™ (Invitrogen-Gibco/LifeTechnologies) system. Multiple gene transfer vectors, such as pAcAB3 andpAcAB4 are particularly preferred.

Favorable baculovirus expression vectors utilize double recombinationbetween the multiple gene transfer vector and baculovirus genomic DNAwhich is linearized by Bsu36.I such that a portion of the essential geneORF1629 is lost due to genomic DNA digestion. Double recombinationbetween the transfer vector and the linearized baculovirus DNA restoresORF1629 and concurrently mediates integration of the heterologous geneexpression cassette into the baculovirus genome. A further favorablefeature of this system is the utilization of baculovirus genomic DNAderived from a baculovirus strain carrying a conditionally-lethal genewhich is lost via linearization with Bsu36.I. This feature ensures thatany contaminating uncut parental baculovirus DNA does not lead to rescueof parental virus, ensuring that all rescued baculoviruses are derivedfrom recombination with the transfer vector.

The various components of the immunogenic composition will be selectedto be pharmaceutically acceptable. As used herein, the term“pharmaceutically acceptable” means not biologically or otherwiseundesirable, in that it can be administered to a subject withoutexcessive toxicity, irritation, or allergic response, and does not causeunacceptable biological effects or interact in a deleterious manner withany of the other components of the composition in which it is contained.A pharmaceutically-acceptable carrier would naturally be selected tominimize any degradation of the baculovirus, insect cells, and othercomponents and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.Pharmaceutically-acceptable ingredients include those acceptable forveterinary use as well as human pharmaceutical use, and will depend onthe route of administration.

Native/wild-type NA proteins are recombinantly expressed at high levelsin the insect cells infected with the baculovirus expression vectors.Advantageously, the primary gene product is unprocessed, full length NAwhich remains associated with the cell membrane of infected insectcells, as well as budded baculovirus membranes. As such, unlike purifiedor soluble forms of NA, such as those which have been modified ormutated, the NA in the inventive compositions is presented on thesurface of such membranes and complexes, likely contributing to theimmunogenicity of the composition (particularly as displayed on thebaculovirus viral membrane surface) as discussed herein.

Expression plasmids can be generated using sequence information for NA,based upon publicly available sequences and/or based upon newlysequenced circulating strains. Exemplary NA sequences include Genbankaccessions KY115564 and KU752376, or conservatively modified variantsthereof that retain the functionality of NA, or sequences which encodefor NA protein, such as Genbank accessions APG56794.1 and AMP44884.1, orconservatively modified variants thereof that retain the functionalityof NA. It will be appreciated that wild-type/native NA sequences for useas vaccines in various species can be identified from publicly availablesequences and/or from circulating strains, such that up-to-date vaccinescan be developed. The identified sequences are synthesized into cDNA andinserted or cloned into the baculovirus expression vectors along withappropriate control sequences, promoters, etc. The resulting expressionvectors can then be transfected into appropriate insect cells, and thecrude culture can be used for vaccine production.

Ideally, the immunogenic compositions will include a mixture of two ormore NA protein subtypes to provide multi-valent protection. However,this will also depend on the species to be vaccinated. For example, pigsare known to be infected with both N1 and N2 strains. However, dogs aremore likely to be infected with only N2. Thus, for certain species, onlyone NA protein subtype may be necessary to achieve vaccination. Theplatform developed is exemplified in pigs in the working examples, wherewe have developed the BacNA1 expression construct that was used in ourstudies. NA1 (SEQ ID NO:2) is derived from A/swine/Iowa/A01782229/2016H1N1 (SEQ ID NO:1). We also generated a BacNA2 expression plasmidderived from A/swine/Oklahoma/A01730659/2016 H1N2 (SEQ ID NO:3). Asnoted above, suitable baculovirus expression shuttle vectors, such aspAcAB3, are commercially available. However, it will be appreciated thatthe vaccine platform can be applied to a variety of other speciessusceptible to influenza infection, including poultry, canines, equines,and felines, as well as humans.

In one or more embodiments concerned with research and study, abaculovirus secretion signal sequence (e.g., GP67) and affinity tag(e.g., polyhistidine-tag) can be added to the NA gene during synthesisto facilitate secretion of the protein into the supernatant during cellculture (which is useful for study and research of the secreted proteinby affinity chromatography). Isolation and purification from culturemedium for research and study is considerably easier than purificationfrom cell lysates, as cellular material does not need to be removed fromthe preparation.

However, for vaccine preparation, baculoviruses expressing native NAproteins can be prepared by gently mix plasmid DNA (e.g., pAcAB3 shuttlevector containing NA1 or NA2) and linearized baculovirus DNA (purchasedfrom ABVECTOR) followed by adding Profectin (ABVECTOR) dropwise into themixture. After a 10-minute incubation, DNA-Profectin mixture will beadded into semi-confluent monolayer of insect cells (e.g., SF9) for anadditional 72-hours of incubation. Following the confirmation of NA1 andNA2 protein expression in Western-blot and NA activity assays, seedvirus stocks (BacNA1 and NacNA1) can be titrated and optimized in termsof the infectious dose and culture time. The cell culture can beinactivated using an appropriate chemical treatment, such as withformaldehyde, β-propiolactone, ethylenimine, binary ethylenimine, orthimerosal (and preferably binary ethylenimine).

Preferably, the antigenic influenza components in the immunogeniccompositions according to the invention consist of influenza NA-typeproteins. That is, the immunogenic compositions are preferablysubstantially free of other influenza proteins, subunits, particles,etc., such as influenza HA, matrix proteins (M1 or M2), RNA polymerasesubunits PB1, PB2, and PA, nucleoprotein (NP), nonstructural proteins(NS1, NS2), or associated virus like particles. As used here,“substantially free” means that the component is not intentionally addedor part of the composition, although it is recognized that residual orincidental amounts or impurities may be present in low amounts (e.g.,less than about 0.1% by weight and preferably less than about 0.01% byweight, based upon the total weight of the composite taken as 100% byweight). That is, the only influenza antigenic component(s) present inthe inventive compositions is NA (and preferably mixtures of NA1 andNA2). More preferably, the recombinantly-expressed NA proteins in thevaccine are membrane-bound or membrane-associated, full-length, ornative NA proteins (as opposed to free, soluble, modified NA proteins).

The immunogenic compositions can comprise a therapeutically effectiveamount of NA dispersed in a suitable carrier. Examples include aqueoussolutions such as sterile water/distilled autoclaved water (DAW),phosphate buffered saline (PBS), normal (n.) saline (˜0.9% NaCl),aqueous dextrose solutions, aqueous glycerol solutions, ethanol, normalallantoic fluid, various oil-in-water or water-in-oil emulsions, as wellas dimethyl sulfoxide (DMSO) or other acceptable vehicles, and the like.In the inventive compositions suitable carriers further include cellculture media used for culturing the insect cells, and cell culturesupernatants. As discussed herein, the expressed NA is preferably notpurified or isolated from the expression systems and mixed with asuitable carrier to generate the inventive vaccines. Rather, theexpressed NA, along with infected insect cells, membrane particles, andrecombinant baculovirus (and associated cell culture and supernatants)can be directly dispersed in a carrier system noted above foradministration without purification. The amount included in thecomposition is an amount that provides a therapeutically effectiveamount of expressed NA. Methods are described herein for detecting theamount of NA expressed from a given expression system.

As used herein, a “therapeutically effective” amount refers to theamount that will elicit the biological or medical response of a tissue,system, or subject that is being sought by a researcher or clinician,and in particular elicit some desired protective effect as against theviral infection by priming or stimulating an immune response specificfor one or more strains of influenza virus (and preferably at least thetarget strain). One of skill in the art recognizes that an amount may beconsidered therapeutically “effective” even if the condition is nottotally eradicated or prevented, but it or its symptoms and/or effectsare improved or alleviated partially in the subject. In someembodiments, the composition will comprise at least about 1 μg total NA,preferably at least 5 μg total NA, more preferably at least 10 μg totalNA, more preferably at least 15 μg total NA, more preferably morepreferably at least 20 μg total NA, and even more preferably about 25 μgper dose or more. As used here “total” NA clarifies that if twodifferent subtypes of NA are included in the vaccine, the total amountof all subtypes of NA in the composition.

Other ingredients may be included in the composition, such as adjuvants,other active agents, preservatives, buffering agents, salts, otherpharmaceutically-acceptable ingredients. The term “adjuvant” is usedherein to refer to substances that have immunopotentiating effects andare added to or co-formulated in the vaccine composition in order toenhance, elicit, and/or modulate the innate, humoral, and/orcell-mediated immune response against the vaccine components. Suitableadjuvants include: aluminum salts, such as aluminum hydroxide, aluminumphosphate, alum (potassium aluminum sulfate), or mixed aluminum salts,peptides, oil or hydrocarbon emulsions, or any other adjuvant deemedsuitable for human or animal use. In one or more embodiments, thebaculoviral components of the expression system itself contributes anadjuvant effect. Antibiotics can also be used as part of vaccineproduction and may be present in small amounts in the vaccine, such asneomycin, polymyxin B, streptomycin and gentamicin.

Advantageously, the immunogenic compositions of the inventionspecifically generate in the subject anti-influenza antibodies againstNA only. Anti-NA antibodies do not prevent virus uptake but ratherprevent the newly formed virus from escaping the cell. This is animportant distinction as our vaccine also generates infection immunity,i.e., it allows a permissive infection by influenza but controlsclinical disease and observable symptoms by preventing virus spread. Notonly do we get immunity targeted at the antigen in the vaccine, we alsostimulate cell mediated immunity by allowing the virus limitedreplication in the vaccinated subject.

Further, maternal anti-HA antibodies are nearly universally present inyoung animals, such as pigs, and interfere with vaccination. Incontrast, anti-NA antibodies are largely low or non-existent in animalsvaccinated with traditional vaccines or following infection. As anti-NAantibodies are low in the animals, we can vaccinate young and growinganimals using the immunogenic compositions and not have maternalantibody interference.

Further, the immunogenic compositions rely on expression of full-length,wild-type/native, membrane-bound NA, which promotes a more robust immuneresponse as compared to purified or soluble versions of NA that havebeen previously attempted. Again, the NA used in the presentcompositions is not processed or purified. Consequently, the NA is foundintegrated in the insect cell membranes and the budded baculovirusmembranes in the culture supernatants. Presentation of NA in thesecomplex structures likely makes it more immunogenic (especially asdisplayed on the baculovirus viral membrane). Measures of NA activity inthe culture have shown that about half is in the cells and the otherhalf is in the supernatant (presumably in the baculovirus membranes inthe supernatant).

Homologous and heterologous challenge studies demonstrate that crudebaculovirus culture (Sf9 cells+baculovirus+Sf900II media) containing atleast 25 g each of NA1 and NA2 yields protection to influenza virus. Theprimary measurement of protection is reduction in lung lesions. Thesecondary measurement of protection is reduction in influenza viraltiter in the lung. Vaccination/challenge studies demonstrate the NAinhibiting antibody titers are a good correlate of protection. Vaccinesformulated at a minimum of 25 μg total NA/dose yield NI titers >40,however, doses as low as 10 μg total NA have been demonstrated toprovide a protective immune response.

Thus, described herein are vaccination methods or methods of stimulatingan immune response against influenza infection, so as to inhibit,reduce, or even prevent symptoms of infection. The methods generallyinvolve administering the immunogenic compositions to a host animalsusceptible to influenza in an effective amount. The composition can bedelivered intramuscularly, subcutaneously, intradermally, orintravenously using a needle and syringe, or a needleless injectiondevice, as well as mucosally, such as intranasal administration.Advantageously, there have been no adverse injection site reactionsobserved in vaccinated subjects thus far. While stimulation of aprotective immune response with a single dose is preferred, additionaldosages can be administered, by the same or different route, to achievethe desired prophylactic or therapeutic effect. The vaccine can also beadministered using a prime and boost regime if deemed necessary. In someembodiments, the methods described herein are useful for eliciting animmune response against influenza infection, as described above.

Advantageously, the compositions provide an immune response andheterologous protection against influenza. Ideally, the compositionsinclude at least one NA subtype NA1 and at least one NA subtype NA2 tobroaden its protection coverage. Such an “immune response” includes, forexample, the production or activation of antibodies, B cells and/or thevarious T cells, directed specifically to NA1 and/or NA2. The immuneresponse will be demonstrated by a lack of observable clinical symptoms,or reduction of clinical symptoms normally displayed by an infectedanimal, faster recovery times from infection, reduced duration or amountof viral shedding, and the like. Accordingly, vaccinated animals willdisplay resistance to new infection (or observable signs of infection)or reduced severity of infection, as compared to unvaccinated animals.“Reducing” the incidence, severity, and/or duration of clinical symptomsand/or viral shedding, means reducing the number of infected animals ina group, reducing or eliminating the number of animals exhibitingclinical signs of infection, or reducing the severity of any clinicalsigns that are present in the animals, in comparison to wild-typeinfection in unvaccinated animals.

In some embodiments, the vaccine can be provided in unit dosage form ina suitable container. The term “unit dosage form” refers to a physicallydiscrete unit suitable as a unitary dosage for human or animal use. Eachunit dosage form may contain a predetermined amount of the vaccine(and/or other active agents) in the carrier calculated to produce thedesired effect. In other embodiments, the vaccine can be providedseparate from the carrier (e.g., in its own vial, ampule, sachet, orother suitable container) for on-site mixing before administration to asubject. A kit comprising the vaccine is also disclosed herein. The kitfurther comprises instructions for administering the vaccine to asubject. The virus can be provided as part of a dosage unit, alreadydispersed in a pharmaceutically-acceptable carrier (e.g., along withculture media and/or adjuvant solution), or it can be providedseparately from the carrier. The kit can further comprise instructionsfor preparing the virus for administration to a subject, including forexample, instructions for dispersing the virus in a suitable carrier.

Using the methodology and technology described herein compositions canbe developed that are effective against H1N1, H3N1, H1N2, and H3N2viruses in pigs, and can be considered “universal” vaccine candidates.In other species, NA subtypes can be sequenced and used to createcompositions that provide protection against various H_(X)N₁ or H_(X)N₂viruses. Specifically, avian influenza is typically H5N1 or H5N2.Chickens or turkeys can be vaccinated with BacNA1/BacNA2 to generate anantibody response to H5N1/H5N2 viruses. Canine influenza is typicallyH3N8 or H3N2 so could predict BacNA2 would protect against canineinfluenza. Likewise, humans are typically infected with H1N1 or H3N2 andmay benefit from the vaccination strategies described herein.

As used herein, the term “vaccine” refers to an immunogenic compositioncapable of eliciting partial or complete immunogenic protection againsta disease or condition in the subject to which it has been administered.Although vaccines are generally considered prophylactic, the vaccinesmay be used for therapeutic treatment of a disease or a condition.Compositions according to the embodiments disclosed herein are useful intreating viral infection from influenza in a subject (e.g., swine)and/or preventing or reducing clinical symptoms of infection. Suchclinical symptoms include respiratory distress, fever, anorexia andlethargy. Thus, embodiments described herein have therapeutic and/orprophylactic uses, and in particular can be used for prophylactictreatment of a viral infection. In general, the compositions areadministered prophylactically, that is, before the subject demonstratesdetectable clinical signs of an infection, such that the subjectdevelops an adaptive immune response to infection by the virus. As such,the methods are useful for preventing the development of observableclinical symptoms from viral infection, and/or reducing the incidence orseverity of clinical symptoms, and/or effects of the infection, and/orreducing the duration of the infection/symptoms/effects, and/or reducingthe amount and/or duration viral shedding/viremia, as compared withunvaccinated control animals. Thus, the composition may only partiallyprevent and/or lessen the extent of morbidity due to the viral infection(i.e., reduce the severity of the symptoms and/or effects of theinfection, and/or reduce the duration of theinfection/symptoms/effects), as compared with unvaccinated controlanimals. Yet, the composition is still considered to treat or “prevent”the target infection or disease, even though it is not 100% effective.

Additional advantages of the various embodiments of the invention willbe apparent to those skilled in the art upon review of the disclosureherein and the working examples below. It will be appreciated that thevarious embodiments described herein are not necessarily mutuallyexclusive unless otherwise indicated herein. For example, a featuredescribed or depicted in one embodiment may also be included in otherembodiments, but is not necessarily included. Thus, the presentinvention encompasses a variety of combinations and/or integrations ofthe specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or moreitems, means that any one of the listed items can be employed by itselfor any combination of two or more of the listed items can be employed.For example, if a composition is described as containing or excludingcomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certainparameters relating to various embodiments of the invention. It shouldbe understood that when numerical ranges are provided, such ranges areto be construed as providing literal support for claim limitations thatonly recite the lower value of the range as well as claim limitationsthat only recite the upper value of the range. For example, a disclosednumerical range of about 10 to about 100 provides literal support for aclaim reciting “greater than about 10” (with no upper bounds) and aclaim reciting “less than about 100” (with no lower bounds).

EXAMPLES

The following examples set forth methods in accordance with theinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Example 1: Construction of a Baculovirus Strains that Expresses SIV NASubtype 1 (NA1) or Subtype 2 (NA2)

The synthetic NA genes were synthesized at Integrated DNA Technologieswith the addition of BamHI sites immediately upstream and downstream ofthe gene sequence. The NA protein genes of A/swine/Iowa/A01782229/2016(H1N1) (SEQ ID NO:1) and A/swine/Oklahoma/A01730659/2016 (H1N2) (SEQ IDNO:3) were downloaded from Genbank under accessions KY115564 andKU752376, respectively.

The synthetic NA genes were then cloned into the BamHI site of E. colipAcAB3 (AB Vector-Catalog Number B2). After transformation into E. coli,DH5α cells, ampicillin-resistant colonies were selected and expanded inovernight 2 mL cultures of Luria-Bertani broth with 100 μg/mLampicillin. Following plasmid DNA isolation with a Qiagen miniprep kit,plasmids were screened by restriction digestion to identify clones whichcontained NA1 or NA2 genes in the proper orientation (pAcAB3-NA1 andpAcAB3-NA2, respectively) by restriction endonuclease digestion. Theidentified clones were further verified by DNA sequencing.

Plasmid DNA (pAcAB3-NA1 and pAcAB3-NA2) along with Bsu36I-linearizedbaculovirus vector DNA (ProEasy, AB Vector, Catalog Number A10) wereused to transfect Sf9 (Spodoptera frugiperda) insect cells to producerecombinant baculovirus encoding either the NA1 (SEQ ID NO:2) or NA2(SEQ ID NO:4) expression cassette. Transfection was performed in a6-well plate with 1×10⁶ cells/well. For each transfection sample,plasmid DNA and Profectin Reagent (AB Vector, Catalog Number T10)complexes were prepared in 12×75 mm sterile tubes as follows:

-   -   Solution A: 45 μl water+5.0 μl Profectin transfection reagent    -   Solution B: 44 μl water+0.1 μg plasmid (pAcAB3-NA1 or        pAcAB3-NA2)+5 μL linearized baculovirus DNA (ProEasy, AB Vector)

Solution A and Solution B were combined and incubated for 20 minutes at20° C.+/−5° C. Then, 0.9 mL of SF-900 II medium was added to each tubebefore adding the complexes into Sf9 cells. The DNA profectin complexeswere transferred into Sf9 cells and incubated for 24 hrs in 270+/−1° C.Two mL of fresh cell culture medium was then added to each well. Thetransfected cells were incubated at 27° C. for generation of recombinantbaculovirus.

Recombinant baculoviruses produced from transfection of Sf9 cells areusually released into the medium at 4 to 5 days post-transfection. Oncethe cells appeared infected (cell enlargement, internal granularformation, cell detached from cell culture surface, cell degradation),the viruses were harvested from the cell culture medium bycentrifugation at 500×g for 10 minutes to remove cells and large debris.The recombinant baculoviruses were called P0 viral stock.

The P1 viral stocks were amplified in Sf9 cells by inoculation of 0.5 mLof P0 material to a T75 shaker flask of ˜60% confluent Sf9 cells in 20mL of Sf900II media. P1 was harvested on day 4 post infection.Similarly, P1 harvests (1 mL) were used to infect 2-T225 flasks of Sf9cells (˜60% confluency) in 100 mL of Sf900II. Passage 2 (p2) recombinantbaculoviruses were harvested on day 4 post infection and aliquoted asthe Master Seed Stock, identified as BacNA1 P2 072817 and BacNA2 P2072817.

Example 2: Verification of NA Activity in BacNA1 and BacNA2

The mature influenza NA protein is a tetrameric protein composed of fouridentical subunits. Mature NA has sialidase activity, cleaving sialicacid moieties on glycoproteins. Only mature, tetrameric protein has thisenzymatic activity. To verify that the NA1 and NA2 genes expressed inbaculovirus are enzymatically active, NA activity assays were performed.

NA activity is assayed using 2-o-(p-nitrophenyl)-α-D-N-acetylneuraminicacid (NP-NANA, SigmaAldrich N1516). For NA activity determination, 50 μLof 0.3 mM NP-NANA is combined with 50 μL of clarified culturesupernatant and incubated in a flat-bottomed 96 well plastic plate for 1hour at 37° C. before stopping the reaction with the addition of 100 μLof 1.0 N NaOH and measuring absorbance at 405 nm. As shown in FIG. 1 ,NA activity is confirmed by an absorbance >0.4 while supernatant from acontrol baculovirus culture has an absorbance <0.2. The controlbaculovirus culture is generated by culturing the baculovirus controlstrain, BacCon, under similar conditions. BacCon was constructed asdescribed for Bac-NA1 and Bac-NA2 except pAcAB3 was used fortransfection of Sf9 cells. BacCon does not contain a NA gene andestablishes the background absorbance for baculovirus propagated in Sf9cells.

Example 3: Protection Afforded Pigs Vaccinated with BacNA1 to aHeterologous H1N1 Challenge

The experimental vaccine consisted of a crude mixture of Sf9 cellsinfected with the BacNA1 and culture supernatant inactivated with binaryethyleneimine formulated with 15% commercial oil-in-water adjuvant(CA50, Cambridge Technologies). Groups of eight, influenza seronegativethree-week old pigs, acquired from Midwest Research Swine, werevaccinated with either inactivated BacNA1 culture fluids, an inactivatedwhole-virus H1N2 SIV or mock vaccinated. Vaccines were administeredintramuscularly at four and six weeks of age. Pigs were challengedintranasally with 2 mL of 6.0 TCID₅₀/mL of a heterologous SIVA/swine/Minnesota/2073/2008 (H1N1). The challenge virus (α cluster) washeterologous both to the inactivated H1N2 vaccine group (γ cluster) aswell as the recombinant NA1 (92% similarity).

Sera collected on the day of challenge was analyzed for α-IAV-Snucleoprotein antibodies using the commercially-available IDEXX ELISAassay. The IDEXX IAV-S NP ELISA and NA inhibition titers in seracollected from pigs on the day of challenge are shown in FIG. 2 . Allpigs inoculated with BacNA1 or mock vaccinated remained negative whilepigs vaccinated with an inactivated H1N2 virus were positive (FIG. 2 ).Sera were also analyzed for NA inhibiting antibody titers using thechallenge virus. Pigs vaccinated with BacNA1 had a mean NI titer of 69while pigs in the other treatment groups were negative (FIG. 2 ).

Nasal swabs were collected on the day of challenge and on days 1, 3 and5 post-challenge. We employed two complementary approaches to detectviral shedding. The first approach was the standard qRT-PCR with theCycle threshold (Ct) values as the readout, which are shown in FIG. 3for Ct values in nasal swabs collected from pigs on days 1, 3 and 5post-challenge. It is generally agreed that the higher Ct values, thelower amount of viral load. The second approach was cell-based TCID₅₀experiment that measures the amount of infectious virus particles innasal swabs. We used swine testicle (ST) cells for determining viralTCID₅₀. All pigs were negative for SIV on the day of challenge byqRT-PCR. Interestingly, we found that our N1-based recombinant vaccineoffered better protection than the inactivated H1N2 vaccine.Specifically, we observed that Cycle threshold (CT) values for pigsvaccinated for BacNA1 were higher, representing lower levels of IAV-Sshedding, on all three days (FIG. 3 ). FIG. 4 shows the SIV titers(TCID50/mL) in nasal swabs collected from pigs on days 1, 3 and 5 postchallenge. Similar to the qRT-PCR results, lower amounts of IAV-S weredetected in BacNA1 vaccinated pigs than pigs vaccinated with inactivatedH1N2 virus or mock vaccinated (FIG. 4 ).

On day 5 post challenge, pigs were euthanized and the lungs were removedin toto and scored by a blinded veterinarian based on gross lungconsolidation in each lobe (%) which was used to calculate totalaffected lung area. FIG. 5 shows the qRT-PCR Ct values and percent lungconsolidation day 5 post challenge. Lung lesions were very low forBacNA1 vaccinated pigs (0.34%) while considerable lung consolidation wasobserved both for H1N2 and mock vaccinated pigs (6.0 and 5.1%,respectively, FIG. 5 ). A portion of the right cardiac lung was alsoanalyzed for SIV by qRT-PCR. SIV was barely detectable for BacNA1vaccinated pigs (Ct=35.4) while high viral loads were detected in theH1N2 and mock treatment groups (Ct's 23.1 and 21.8, respectively).

A portion of the right cardiac lobe was also fixed in formalin andanalyzed for histopathology. Samples were scored on a 0-4 scalerepresenting increased pathology based on hematoxylin and eosin stainingand immunohistochemistry for SIV. Microscopic lung lesion scores basedon hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)for IAV-S are shown in FIG. 6 . The pathologist was blinded to the studydesign. Similar to the gross lung lesions, very little lung pathologywas observed microscopically and SIV was not identified by IHC in BacNA1vaccinated pigs (FIG. 6 ). In contrast, significantly more lung lesionsalong with associated IAV-S detection was observed in the H1N2 and mocktreated pigs.

Example 4: Protection Afforded Pigs Vaccinated with BacNA1 and BacNA2 toa Heterologous H1N1 Challenge

Baculovirus N1 and N2 cultures (two separate strains) were concentrated10× by ultracentrifugation and used to formulate vaccines with straightantigen (0.85 mL N1 10× concentrate, 0.85 mL N2 10× concentrate, 0.3 mLCA50 adjuvant per dose) or 1:5 and 1:20 fold dilutions of the 10×antigen concentrate. Pigs vaccinated at 3 and 5 weeks of age andchallenged at 7 weeks of age with a heterologous H1N1 (6 TCID₅₀/mL)which has a N1 protein ˜92% similar to BacNA1.

FIG. 7 shows the data from serology (NA inhibition assay) using parentalH1N1 and H1N2 viruses as well as the challenge virus showing antibodygenerated to N1 and N2. Serum was collected from all pigs on the day ofchallenge. The NI assay results show that both BacNA1 and BacNA2 areable to stimulate an antibody response which is capable of inhibitingNA1 and NA2 activity. Importantly, antibodies generated against BacNA1are able to cross react with the heterologous virus used for challenge.

FIG. 8 shows the results from PCR on lung tissue collected 5 days postchallenge with heterologous H1N1. These results show a dose-dependenteffect such that influenza virus titers in the lungs post-challenge areinversely correlated with the amount of antigen used for vaccination.

Gross and microscopic lung scores on lungs collected 5 days postchallenge are shown in FIG. 9 . These results demonstrate that all threevaccines formulations were capable of providing protection to lungdamage caused by influenza virus infection. As the antigen pool used togenerate the three vaccines was 10× concentrated baculovirus cultures,the vaccines represented approximately 8.5, 1.7 and 0.43 mL of BacNA1and BacNA2 per dose.

Example 5: Quantification of NA Produced by BacNA1 and BacNA2

The full length native sequence of NA1 and NA2 expressed by BacNA1 andBacNA2, respectively, each contain an N-terminal transmembrane domainand consequently are found associated with Sf9 cellular or baculoviruslipid membranes. Membrane proteins are difficult to purify. In order togenerate highly purified reference standards for NA1 and NA2 weexpressed N-terminal truncated versions of NA1 (SEQ ID NO: 5) and NA2(SEQ ID NO:6) in baculovirus which included the baculovirus GP67secretion signal sequence to enable soluble protein expression. NAprotein genes of A/swine/Iowa/A01782229/2016 (H1N1) andA/swine/Oklahoma/A01730659/2016 (H1N2) were synthesized such that thenucleotides encoding the N-terminal 35 amino acids were removed andreplaced by the baculovirus Gp67 secretion signal peptide and ahexahistidine tag was added at the C-terminus of the protein (SEQ IDNO:5 and SEQ ID NO:6). The resulting genes were cloned into pAcAB3 andused to construct recombinant baculoviruses expressing soluble NA1 andNA2 as described in Example 1. Sf9 cell cultures of the resultingbaculovirus strains were used to purify soluble NA1 and NA2 from theculture supernatant using affinity chromatography (immobilized Ni2+).The purified NA1 and NA2 proteins were visualized by SDS-PAGE andWestern blotting using an antibody which recognizes the hexahistidinetag.

The purity of NA in each preparation was determined by densitometry ofthe SDS-PAGE gel to be >90%. In FIG. 10 , Lane 1 contains 2 ug of bovineserum albumin standard as a reference. Lane 2 contains 2 ug of purifiedNA. Lane 3 contains 2 ug of purified NA. Protein concentration wasdetermined by the Bradford assay using a bovine serum albumin standardcurve. Purified NA1 was 0.21 mg/mL and NA2 was 0.14 mg/mL.

NA activity assays were performed on representative cultures of BacNA1and BacNA1 along with purified, soluble NA1 and NA2 as described inExample 2. Parallel line analysis was performed on the NA activity assaycurves to determine the concentration of NA1 and NA2 produced by BacNA1and BacNA2, respectively, using the purified reference NA1 and NA2 ofknown concentration. Repeated measures of BacNA1 and BacNA2 culturesfound approximately 50 μg/mL NA in both cultures.

Example 6. Protection Afforded Pigs Vaccinated with BacNA1 and BacNA2 toa Homologous H1N2 Challenge

Baculovirus N1 and N2 cultures (two separate strains) were concentrated10× by ultracentrifugation. The amount of NA present in the concentratedcultures were determined as described in Example 5. Vaccines wereformulated to contain 200, 100, 50, 25 or 0 μg/dose of both NA1 and NA2and 15% CA50 adjuvant. The dose volume was adjusted to 2 mL withphosphate buffered saline. Pigs were vaccinated at 3 and 5 weeks of ageand challenged at 7 weeks of age with the homologous H1N2 (6 TCID50/mL)parental virus.

Serology-NA inhibition antibody titer to parental viruses are shown inFIG. 11 . All vaccine formulations induced antibody titers to bothparental viruses. IAV PCR (Ct) values on nasal swabs collected on days1, 2 and 5 post challenge and lung tissue collected day 5 post challengeare shown in FIG. 12 . The amount of virus detected in nasal swabs andlungs were lower than mock vaccinated pigs at all time points. FIG. 13includes the gross and microscopic scores on lung tissue collected day 5post challenge. All vaccine formulations significantly reduced lunglesions.

Example 7. Protection Afforded Pigs Vaccinated with BacNA1 and BacNA2 toa Homologous H1N1 Challenge

Baculovirus N1 and N2 cultures (two separate strains) were concentrated10× by ultracentrifugation. The amount of NA present in the concentratedcultures were determined as described in Example 5. Vaccines wereformulated to contain 200, 100, 50, 25 or 0 μg/dose of both NA1 and NA2and 15% CA50 adjuvant. The dose volume was adjusted to 2 mL withphosphate buffered saline. Pigs were vaccinated at 3 and 5 weeks of ageand challenged at 7 weeks of age with the homologous H1N1 (6 TCID50/mL)parental virus.

Serology-NA inhibition antibody titer to parental viruses are shown inFIG. 14 . All vaccine formulations induced antibody titers to bothparental viruses. The IAV PCR (Ct) values on nasal swabs collected ondays 3 and 5 post challenge and lung tissue collected day 5 postchallenge are shown in FIG. 15 . While the amount of influenza detectedin nasal swabs for vaccinated pigs was not significantly different thanmock vaccinated pigs, all vaccine groups had significantly lower levelsof influenza in lung samples. FIG. 16 shows the lung lesion scores GMT(geometric mean titer) as assessed by H&E and IHC staining weresignificantly reduced in all vaccine groups as compared to mockvaccinated pigs.

Example 8. Dose Titration Studies

Further, dose titration studies were carried out. Pigs were vaccinatedwith crude baculovirus culture fluids as described at various dilutionlevels. The results are shown in FIG. 17-19 . The 1× composition wasformulated with 42.5 μg of NA1 and 42.5 μg of NA2 per dose. For the mostdilute vaccine, 5 μg of NA1 and 5 μg of NA2 were enough antigen to giveprotection. FIG. 17 is a graph of serology using the NA inhibitionassay. It clearly shows seroconversion for all vaccines and a successfuldose response (decreasing antigen leading to lower NI titer). FIG. 18shows the results of lung lesion scores for N1 and N2. FIG. 19 showslung lesion scores as assessed by H&E and IHC staining weresignificantly reduced in all vaccine groups as compared to mockvaccinated pigs. These results demonstrate that the crude baculovirusculture fluids can be diluted ˜8× and still be efficacious (˜0.1 mLBacNA1/BacNA2 per dose).

Discussion

The results from these vaccination trials convincingly demonstrate thatimmunity based on influenza NA-alone provides significant protectionfrom homologous and heterologous challenge. While NA-based immunity doesnot prevent infection, pigs were nearly completely protected from lungdamage. Vaccination aimed at controlling viral dissemination in vivo mayallow for the generation of infection immunity while mitigating clinicaldisease. Another exciting outcome of this study was the ability ofrecombinant NA1 to provide significant protection to a heterologous H1N1challenge.

To date, only SIVs containing two NA subtypes (N1 or N2) infect theglobal swine industry and cause a significant economical concern. The NAgenes of SIVs are less variable than the HA genes, which represents agood vaccine target. Currently FDA-approved inhibitors to treat humaninfluenza are also targeting the functional NA protein. Including a HAcomponent in the vaccines such as the marketed inactivated SIV vaccineswill drive immune responses directed largely towards the immunodominantHA so antibody responses to NA with the universal protection potentialwill be masked or reduced.

The invention claimed is:
 1. An immunogenic composition for inducing animmune response that mitigates clinical disease in an animal frominfluenza after administration, said composition comprising recombinantbaculovirus expression vectors in cultured insect cells dispersed in apharmaceutically-acceptable carrier comprising insect cell culturemedia, optional adjuvant, said recombinant baculovirus expressionvectors expressing a therapeutically effective amount of neuraminidase,said neuraminidase being associated with the cell membrane of saidinsect cells and/or baculovirus viral membrane surfaces in saidcomposition and presented on the surfaces of said membranes, whereinsaid animal is selected from the group consisting of avians, canines,equines, and felines.
 2. The immunogenic composition of claim 1, saidrecombinant baculovirus expression vectors expressing native,full-length neuraminidase protein.
 3. The immunogenic composition ofclaim 1, said recombinant baculovirus expression vectors expressingneuraminidase derived from sequences of H1N1, H3N1, H1N2, H3N2, H5N1,H5N2, H3N8, and/or H3N2.
 4. The immunogenic composition of claim 1, saidcomposition comprising a first set of recombinant baculovirus expressionvectors expressing neuraminidase subtype 1 and a second set ofrecombinant baculovirus expression vectors expressing neuraminidasesubtype
 2. 5. The immunogenic composition of claim 1, wherein theinfluenza antigenic components in said composition consists of influenzaneuraminidase-type proteins.
 6. The immunogenic composition of claim 1,wherein said composition is chemically inactivated.
 7. The immunogeniccomposition of claim 1, wherein said composition is unpurified.
 8. Theimmunogenic composition of claim 1, wherein said insect cells areSpodoptera frugiperda cells, Trichplusia ni cells, Bombyx mori cells, ora cell line derived therefrom.
 9. The immunogenic composition of claim1, wherein said cell culture medium is serum free culture medium. 10.The immunogenic composition of claim 1, said pharmaceutically-acceptablecarrier further comprising phosphate buffered saline.
 11. Theimmunogenic composition of claim 1, in unit dosage form.
 12. Theimmunogenic composition of claim 1, said composition being substantiallyfree of influenza hemagglutinin.
 13. A kit for stimulating a universalimmune response against influenza infection, said kit comprising: animmunogenic composition according to claim 1; and instructions foradministering said composition to a host animal susceptible toinfluenza.
 14. A method of stimulating an immune response in an animalthat mitigates clinical disease from influenza infection, said methodcomprising administering an immunogenic composition according to claim 1to an animal susceptible to influenza in an effective amount, whereinsaid animal is selected from the group consisting of avians, canines,equines, and felines.
 15. The method of claim 14 wherein said effectiveamount comprises a therapeutically effective amount of neuraminidaseexpressed in said composition.
 16. The method of claim 14, saidcomposition being administered intramuscularly, subcutaneously,intradermally, intravenously, or mucosally.
 17. The method of claim 14,said immune response comprising production or activation of antibodies,B cells and/or T cells, directed specifically to neuraminidase in saidanimal.
 18. The method of claim 14, said immune response comprisingproduction or activation of antibodies, B cells and/or T cells, directedto both neuraminidase subtype 1 and subtype 2 in said animal.
 19. Themethod of claim 14, wherein said immunogenic composition provides aprotective immune response in said animal against H1N1, H3N1, H1N2,H3N2, H5N1, H5N2, H3N8, and/or H3N2.
 20. The method of claim 14, whereinsaid animal is a young animal less than 8 weeks of age, wherein maternalantibodies in said young animal do not interfere with said immunogeniccomposition or immune response.